|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Internal Medicine (Hematology),
Yale University School of Medicine, New Haven, CT; and the Puget Sound
Blood Center and the Department of Medicine (Hematology), University of
Washington School of Medicine, Seattle.
Adducins are a family of cytoskeletal proteins encoded by 3 genes
(alpha, beta, and gamma). Platelets express alpha and gamma adducins,
in contrast to red blood cells that express alpha and beta adducins.
During platelet activation with thrombin, calcium ionophore A23187, or
phorbol 12-myristate 13-acetate, alpha and gamma adducins were
phosphorylated by protein kinase C (PKC) as detected by an antibody
specific for a phosphopeptide sequence in the highly conserved carboxy
terminus. Platelet activation also led to adducin proteolysis;
inhibition by calpeptin suggests that the protease was calpain. The
kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin
and also inhibited proteolysis of adducin. Experiments with recombinant
alpha adducin demonstrated that the PKC-phosphorylated form was
proteolyzed at a significantly faster rate than the unphosphorylated
form. The concentration of adducin in platelets was estimated at 6 µM, similar to the concentration of capping protein. Fractionation of
platelets into high-speed supernatant (cytosol) and pellet (membrane
and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the
cytosol during platelet activation. Platelet aggregation detected
turbidometrically was decreased in the presence of staurosporine and
was completely inhibited by calpeptin. Thrombin-induced changes in
morphology were assessed by confocal microscopy with fluorescein
phalloidin and were not prevented by staurosporine or calpeptin. Our
results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.
(Blood. 2002;99:2418-2426) After stimulation with agonists, platelets
undergo a complex set of events described as activation, adhesion (to
endothelial cells), aggregation (to each other), and clot retraction.
Activation includes secretion of granule contents by fusion with the
open canalicular system,1 shape changes such as extension
of filopodia and lamellipodia,2 and changes in the
fibrinogen receptor, integrin One of the major cytoskeletal components of platelets is actin, which
exists in the platelet as monomeric, ie, G-actin, and filamentous, ie,
F-actin. In the resting platelet, most of the actin is in the monomeric
state, and some exists as short actin filaments with capping proteins
preventing polymerization into longer filaments. When platelets are
activated, most of the G-actin polymerizes into F-actin, causing
extensions of the platelet membrane into filopodia and
lamellipodia.6 In addition to the cytoplasmic G- and
F-actin in platelets, there is a membrane skeleton, a specialized structure composed of crosslinked short actin filaments with linkages to integral membrane proteins.7,8 The membrane skeleton
has been most studied in red blood cells in which short actin filaments are crosslinked by spectrin tetramers.9 Platelets also
contain spectrin in their membrane skeleton,10 but a more
abundant component of the platelet membrane skeleton is
actin-binding-protein 280 (ABP-280) or filamin.11
Although many components of the red cell membrane skeleton are
expressed in platelets, platelets also have cytoplasmic actin filaments
and microtubules contributing to their shape. The complex cytoskeleton
of platelets requires careful coordination to regulate physiologic
changes in these structures. We have identified another cytoskeletal
component of platelets, adducin, that may have a role in the
cytoskeletal changes occurring during platelet activation.
Adducin was first described in red blood cells12,13 but is
found in all human cells examined14 and may function in
the cytoplasm as well as on the membrane skeleton. Adducins (alpha, beta, and gamma) are a family of cytoskeleton proteins encoded by 3 distinct genes.15 The functions of adducin purified from red blood cells have been well studied by in vitro assays. Purified red
cell adducin consists of alpha and beta polypeptides tightly associated
into dimers or tetramers.16 The in vitro functions of red
cell adducin (alpha/beta) include cross-linking spectrin and actin
filaments,17 capping actin filaments,18 and
bundling actin filaments.19 Adducin's interaction with
spectrin and actin is regulated via phosphorylation by several protein
kinases20-23 and by calcium/calmodulin.17
Gamma adducin was discovered as a protein kinase C (PKC)-binding
protein by screening a kidney complementary DNA expression library with
PKC as a probe.24,25
In contrast to the alpha/beta adducin complex found in red cells, the
adducin complex found in platelets and most nonerythroid cells consists
of alpha and gamma subunits.14 Platelet adducin (alpha/gamma) may have different properties from the red cell alpha/beta complex, and these properties may be crucial to adducin's function in platelets and other nonerythroid cells. In experiments reported here, we found that alpha and gamma adducins were
phosphorylated at a PKC phosphorylation site upon activation of
platelets by thrombin, calcium ionophore A23187, or phorbol
12-myristate 13-acetate (PMA). PKC phosphorylation of adducin was
associated with a shift of adducin from the high-speed pellet (membrane
and cytoskeleton) to the high-speed supernatant (cytosol). Platelet
activation also was associated with rapid proteolysis of adducin that
was inhibited by the calpain-specific inhibitor calpeptin. In vitro
experiments demonstrated that PKC-phosphorylated adducin was
proteolyzed more rapidly than unphosphorylated adducin.
These results provide groundwork toward understanding a possible role
of adducin in platelet function. Assays of platelet function
demonstrated that platelet aggregation was decreased in the presence of
staurosporine and completely inhibited by calpeptin. Thrombin-induced
platelet shape change (extrusion of filopodia and lamellipodia) was not
prevented by staurosporine or calpeptin. Our results suggest that
regulation of adducin function by PKC and calpain may play a role in
platelet aggregation.
Platelet preparation
Platelet activation
Western blotting and Coomassie blue-stained gels Samples were separated by SDS polyacrylamide gel electrophoresis on 4% to 15% gradient gels (Biorad) at 100 V. The same sample volume was loaded for the corresponding Coomassie blue-stained gels and the gels that were used for the Western blots. Gels were stained for 15 minutes in Coomassie Brilliant Blue R-250 (0.1% Coomassie blue, 50% methanol, 10% acetic acid), followed by destaining (10% methanol, 7% acetic acid). Samples run on duplicate gels were transferred to nitrocellulose electrophoretically and hybridized with antibodies. Nitrocellulose blots were incubated overnight at 4°C in buffer containing phosphate-buffered saline (PBS), 4% bovine serum albumin (BSA; Fraction V; Sigma), 0.1% Triton X-100, and primary antibody. Blots were washed 3 times at room temperature in the same buffer without BSA, then incubated with protein A conjugated to horseradish peroxidase (Biorad) in buffer with BSA for 60 minutes at room temperature. Blots were washed again 4 times, then incubated with chemiluminescence substrate (Amersham, Piscataway, NJ) and exposed to film. The antibodies used were either antiphosphoadducin (rabbit immunoaffinity-purified immunoglobulin G; Upstate Biotechnology, Lake Placid, NY), or a general antiadducin antibody made in our laboratory. The antiadducin antibody was generated in rabbits by using several synthetic adducin peptides conjugated with glutaraldehyde to rabbit serum albumin (Sigma). The rabbit serum was affinity purified on a column of recombinant human alpha adducin, yet it reacts with alpha, beta, and gamma adducins.Recombinant alpha adducin Full-length human alpha adducin was expressed in BL21(DE3) pLysS Escherichia coli by using the pCRT7/NT-TOPO expression vector (Invitrogen, Carlsbad, CA). The vector was constructed so that the amino terminus of alpha adducin is synthesized in frame with a 6-histidine tag. The 6-histidine tag permitted purification of the expressed polypeptide by using immobilized metal affinity chromatography. For purification, the cobalt-containing Talon Superflow Resin (Clontech, Palo Alto, CA) was used. Extraction and elution buffers were prepared according to the user manual for isolation under native conditions. Recombinant adducin was further concentrated by using centriplus (YM-50) centrifuge filters (Millipore, Bedford, MA).Quantitation of adducin in platelets Adducin levels in platelet samples were determined by using optical density measurements from Western blots and reference to known adducin levels in samples of recombinant alpha adducin. The concentration of recombinant alpha adducin was determined by using the Biorad protein assay kit with BSA as standard. Several dilutions of recombinant alpha adducin and platelet samples from 2 individuals were run on a 4% to 15% polyacrylamide gel and transferred to nitrocellulose. Western blotting with the antiadducin antibody was performed as described earlier. Adducin levels in the standards were calibrated to an optical density scale by using light intensity measurements of scanned gel images analyzed by using the public domain National Institutes of Health (NIH) Image program. The amount of alpha adducin in each platelet sample was calculated from the standard curve. The number of platelets was determined by a resistive-particle counter after column purification, and the concentration of alpha adducin per platelet was calculated by dividing the amount of adducin from Western blot by the number of platelets loaded on the gel.Calpain proteolysis of recombinant adducin Recombinant alpha adducin (240 ng/µL) was phosphorylated by incubation in a reaction-containing adenosine triphosphate 1.6 mM, CaCl2 4.2 mM, PMA 100 nmol/L, recombinant PKC 0.83 ng/µL (Upstate Biotechnology), NaCl 0.1 mol/L, EGTA 0.8 mM, and
NaPO4 0.02 M pH 7.4. Unphosphorylated alpha adducin was
prepared by incubating in the same reaction mix with water replacing
PKC. Mixes were incubated for 30 minutes in a 30°C water bath. After the incubation at 30°C, samples were divided into 1.5-mL
microcentrifuge tubes for the time course of calpain proteolysis.
Twenty microliters of human erythrocyte Calpain I (diluted to 0.003 U/µL; Calbiochem) was added to each tube containing 9.6 µg alpha
adducin (PKC phosphorylated or unphosphorylated), and samples were
incubated for different lengths of time, ranging from 15 to 60 seconds.
The reactions were stopped with 20 µL Laemmli sample buffer and
boiled for 10 minutes. Samples were analyzed by Western blotting, and
relative intensity of bands was calculated from the optical density of bands determined from scanned gel images by using NIH Image software. For each time point, the amount of adducin remaining was calculated from relative intensity and plotted as a curve. P for the
difference between the slopes of the curves was determined by Tukey
studentized range test.
Platelet fractionation Column-purified platelets were divided into 1.5-mL Beckman microfuge tubes at 37°C (900 µL per tube). Thrombin (0.1 U/mL or 1 U/mL) and CaCl2 (2.5 mM) were added, and samples were inverted 6 times and kept at 37°C. Some samples were preincubated with calpeptin (200 µg/mL) for 30 minutes. At indicated time points after activation, 100 µL 10× concentrated lysis buffer was added (final concentration 1% Triton X-100, 2.5 mM EGTA, 4 mM Pefabloc-[Roche, Indianapolis, IN], 0.01 mg/mL Aprotinin [Sigma], 10 mM sodium orthovanadate [Calbiochem], 0.01 mg/mL Leupeptin [Sigma], 1 mM phenylmethylsulfonyl fluoride [Sigma]). Samples were vortexed and put on ice for 20 minutes. Samples were then centrifuged in a Beckman TLA 100.4 ultracentrifuge at 55 000 rpm (126 203g) for 3 hours. Gel samples were made by adding 400 µL Laemmli sample buffer to 800 µL supernatant and resuspending the pellet with 750 µL Laemmli sample buffer. Samples were boiled for 10 minutes and loaded proportionately (45 µL supernatant to 22.5 µL pellet) onto 4% to 15% gradient polyacrylamide gels as described earlier. Western blotting was performed as described above, and relative optical density was determined for adducin and PKC-phosphorylated adducin by using NIH Image software.Platelet aggregation assay Column-purified platelets were assayed by using a 4-channel platelet aggregation chromogenic kinetic system (PACKS-4; Helena Laboratories, Beaumont, TX) with constant stirring (1000 rpm) at 37°C. Platelets were incubated with 0.3 µM staurosporine, 200 µg/mL calpeptin, or 0.8% DMSO (vehicle control) for 30 minutes before activation. After 30 minutes, samples were added to siliconized glass tubes with a stir bar and activated with 1 U/mL thrombin in the presence of 2.5 mM CaCl2. Maximum aggregation within 2 minutes was determined by the aggregometer and averaged for 3 experiments. P was determined by Tukey studentized range test.Fluorescence microscopy Platelets were prepared by column purification as described above. Duplicate samples were made for Western blotting and for microscopy. After incubation with the inhibitor staurosporine (0.3 µM) or calpeptin (200 µg/mL) for 30 minutes or none, platelets were activated with thrombin for 60 seconds. Activation was stopped by addition of glutaraldehyde (final concentration 2%). AlexaFluor 488 phalloidin (Molecular Probes, Eugene, OR) was added to each sample (final concentration 0.002 U/µL phalloidin), and samples were kept at 4°C overnight or longer. On the day of microscopy, samples were pelleted in a microfuge at 10 000 rpm (6720g) for 5 minutes. After the supernatant was removed, the pellet was resuspended with 1 mL PBS, vortexed, and spun again. Pellets were resuspended in 200 µL PBS, and aliquots were placed on glass slides with a coverslip for examination with a Leica confocal microscope.
Figure 1 shows a Coomassie
blue-stained gel (A) and Western blots (B,C) of platelets activated
with 0.1 U/mL thrombin for increasing amounts of time (10 seconds to 5 minutes). For this experiment, a dilute concentration of thrombin was
used to capture the state of phosphorylated adducin with minimal
proteolysis. Standard doses of thrombin (1 U/mL) caused rapid
proteolysis of adducin, making it difficult to assess the level of
phosphorylation (see below). As a control for the degree of platelet
activation, arrows in panel A indicate the positions of ABP-280 and
talin, cytoskeletal components that also undergo proteolysis during
platelet activation.27 Although immunoblotting would be
necessary to assess minor proteolysis of ABP-280 or talin, panel A
shows that dilute thrombin did not cause major proteolysis of ABP-280
or talin (compare with figures below).
The antibody used in panel B is specific for the PKC-phosphorylated form of adducin. This antibody was raised against the phosphopeptide CKKFRTP[pS]FLKKNK, corresponding to amino acids 656-668 of human gamma adducin (Upstate Biotechnologies). The serine residue in this site is phosphorylated by PKC and is conserved among alpha, beta, and gamma adducins.15 In resting platelets (lane 0), there was a low level of PKC phosphorylation of adducin, but, within 10 seconds of addition of thrombin, there was a large increase in the level of PKC phosphorylation of adducin. The upper band, migrating at approximately 125 kDa is alpha adducin, and the 2 lower bands are alternatively spliced isoforms of gamma adducin (80 and 90 kDa) (for review see Suriyapperuma et al15). Beta adducin is not expressed in platelets.14 The antibody used in panel C recognizes alpha and gamma adducins independent of their state of phosphorylation. This antibody was used as a control to demonstrate that adducin was present (but relatively unphosphorylated) in the resting platelets, lane 0. In addition, the general adducin antibody demonstrated that the amount of adducin decreased with time of exposure to thrombin and started as quickly as 10 seconds. The intensity of the thrombin-induced PKC-phosphorylated bands appeared constant from 10 seconds to 5 minutes (panel B), yet the decreased amount of total adducin from 10 seconds to 5 minutes (panel C) would suggest that there is an increased proportion of phosphorylated adducin with time of exposure to thrombin. From this experiment it is not possible to determine whether the adducin being proteolyzed is phosphorylated or not; the PKC-phosphorylated adducin could be preferentially proteolyzed while PKC phosphorylation of unphosphorylated adducin would continue, giving a net result that the level of phosphorylated adducin would appear unchanged. It should be noted that if platelets were prepared without prostacyclin in the column buffers, then the adducin was already PKC phosphorylated before addition of thrombin, and a lower yield of platelets was obtained (data not shown). Activation of PKC is a well-studied component of thrombin-induced
platelet activation.28-30 To verify that the
phosphorylation of adducin detected with the antiphosphoadducin
antibody was due to the action of PKC, platelets were stimulated with
PMA that directly activates PKC. Figure 2
shows a comparison of thrombin activation (1 U/mL) versus activation
with PMA (100 nmol/L). In panel B, the phosphospecific antibody
demonstrated very low levels of adducin phosphorylation in resting
platelets (lane 1) and increased adducin phosphorylation with exposure
to thrombin (lanes 2 and 3) or PMA (lanes 4-6). In panel C, the general
adducin antibody again demonstrated a large amount of adducin in
resting platelets (lane 1) and decreased adducin in both thrombin and
PMA-activated platelets (lanes 2-6). These results confirm that the
thrombin-induced phosphorylation of adducin detected with this antibody
was due to the action of PKC.
To characterize the calcium dependence of the adducin phosphorylation
and proteolysis, platelets were activated with the calcium ionophore
A23187. Figure 3 shows platelet
activation by using increasing concentrations of calcium ionophore
A23187 (0.1 to 4 µM for 2 minutes). Panel A shows that extensive
proteolysis of ABP-280 and talin occurred at higher concentrations of
A23187 (1-4 µM). This proteolysis was blocked by preincubation for 5 minutes with 5 mM EGTA to chelate extracellular calcium before the
exposure to A23187 and calcium (data not shown).
In panel B, phosphorylation of adducin was seen at lower concentrations of A23187 (0.1 and 1 µM), but at higher concentrations (2 and 4 µM) less phosphorylation was seen because the adducin was already proteolyzed (panel C). At 0.1 µM A23187, adducin was phosphorylated by PKC but not yet proteolyzed, suggesting that PKC phosphorylation precedes proteolysis of adducin in platelets. At lower concentrations of A23187 (1-50 nmol/L), PKC phosphorylation of adducin was not detected (data not shown). EGTA blocked both the phosphorylation and the proteolysis of adducin (data not shown). These results suggest that the phosphorylation of adducin is calcium dependent, which confirms the results above, suggesting that PKC phosphorylated adducin during platelet activation. Platelets contain a calcium-dependent protease, calpain, that is known
to proteolyze the cytoskeletal proteins ABP-280, talin, spectrin, and
cortactin10,27,31 as well as the integrin
The observation of rapid PKC phosphorylation and calpain proteolysis of
adducin during platelet activation suggested that PKC phosphorylation
might promote the proteolysis of adducin. To test this possibility, we
preincubated platelets with a kinase inhibitor to determine whether
blocking phosphorylation of adducin would also block proteolysis of
adducin. Figure 5 shows the results of
preincubating platelets with staurosporine (0.1 and 0.3 µM)29 before activation with thrombin. Panel B shows the
typical PKC phosphorylation of adducin with addition of thrombin (lanes
2 and 3) but no PKC phosphorylation of adducin in platelets
preincubated with staurosporine before addition of thrombin
(lanes 5-8). Lane 1 shows resting platelets, and lane 4 shows platelets
preincubated with staurosporine and not activated with thrombin. Panel
C, using the general adducin antibody, shows the typical proteolysis of adducin in thrombin-activated platelets (lanes 2 and 3) but no proteolysis in the platelets pretreated with staurosporine before thrombin activation (lanes 5-8). These results suggest that proteolysis of adducin could be dependent on PKC phosphorylation of adducin. It is
also possible that staurosporine inhibited proteolysis of adducin by
indirectly inhibiting the activation of calpain.
To compare adducin with other actin-binding proteins in platelets and
to better understand its possible role in platelet activation, we
quantitated the amount of adducin in platelets. Recombinant alpha
adducin was used to generate a standard curve for analysis of the
concentration of adducin in platelets by Western blot. Figure
6A shows a Western blot with whole
platelet extracts from 2 individuals (lanes marked X) and 5 dilutions
of recombinant alpha adducin (lanes 1-5). Panel B shows the standard
curve derived from this Western blot and the positions of the 2 platelet samples (triangles) on the curve. The amount of adducin per
platelet based on these 2 samples was determined as
3.5 × 10
To test our hypothesis that PKC phosphorylation of adducin promotes
proteolysis by calpain, we examined calpain proteolysis of recombinant
alpha adducin with and without phosphorylation by PKC. Figure
7 shows the results of a typical
experiment in panels A (anti-adducin Western blot) and B
(anti-phosphoadducin Western blot). PKC-phosphorylated adducin
decreased to a level of 12% after 60 seconds of incubation with
calpain I, whereas unphosphorylated adducin decreased to a level of
58% after 60 seconds of incubation with calpain I (average of 3 experiments quantitated in panel C). On very long exposures, there was
a band corresponding to an intermediate product of proteolysis at about 55 kDa, but this band was also proteolyzed over time, confirming our
results with platelets that stable intermediate products of proteolysis
are not seen after platelet activation. On Coomassie blue-stained
gels, the disappearance of the 125 kDa alpha adducin band confirmed
that adducin was proteolyzed by calpain and not just undergoing a
change in conformation, making it no longer recognizable by antibodies.
Quantitation of calpain proteolysis shown in panel C demonstrated that
the rate of proteolysis of PKC-phosphorylated adducin was faster than
the rate of proteolysis of unphosphorylated adducin. The difference
between the slopes of the 2 curves was statistically significant at
P < .001.
PKC phosphorylation of adducin has been shown to be associated with a
shift of adducin from the membrane skeleton to the cytosol in MDCK
cells treated with PMA.35 To determine whether the PKC phosphorylation of adducin in platelets was associated with a shift in
subcellular location, we fractionated platelets into high-speed
(126 000g × 3 hours) supernatant and pellet fractions at
time points after addition of dilute thrombin (0.1 U/mL). Western blots
were performed on supernatant and pellet fractions, and the optical
density of adducin and PKC-phosphoadducin bands were quantitated with
NIH Image. Figure 8A shows increased
adducin in the supernatant and decreased adducin in the pellet versus time of exposure to thrombin. Although the amount of adducin in the
pellet continued to decrease with time, the amount in the supernatant
remained constant, so that total adducin decreased over time,
consistent with proteolysis. Figure 8B shows the amount of
PKC-phosphorylated adducin in supernatant versus pellet fractions (thrombin 0.1 U/mL, n = 2). There was a rapid increase of
PKC-phosphorylated adducin in the supernatant fraction (10 seconds),
followed by a decrease consistent with proteolysis.
To examine the effect of PKC phosphorylation of adducin without proteolysis, platelets were preincubated with calpeptin (200 µg/mL) before thrombin activation (1 U/mL, n = 2). Figure 8C shows that PKC-phosphorylated adducin in the supernatant continued to increase over time in contrast to the decrease seen without calpeptin (Figure 8B). PKC-phosphorylated adducin in the pellet also increased up to the 30-second time point and then decreased, consistent with movement of phosphoadducin to the supernatant. Together, these data suggest an association between PKC phosphorylation of adducin and movement to the cytosol during thrombin activation of platelets. To determine how adducin phosphorylation by PKC and proteolysis by
calpain may be involved in platelet function, platelet aggregation was
assayed by using a 4-channel aggregometer. Four samples were tested
simultaneously: untreated control, staurosporine-treated, calpeptin-treated, or DMSO-treated control. Thrombin (1 U/mL) was used
to stimulate aggregation in the presence of 2.5 mM CaCl2. Results of a typical experiment are shown in Figure
9. Untreated platelets (A) and
DMSO-treated control platelets (B) showed no difference and aggregated
within 2 minutes to a maximum of 77% ± 0.8% and 76.8% ± 1.7%
(mean of 3 experiments). Aggregation of staurosporine-treated platelets
(C) was inhibited to a maximum of 64.1% ± 0.5%; the difference
from control was statistically significant (P = .003).
Aggregation of calpeptin-treated platelets (D) was completely inhibited
with a maximum of 3.8% ± 1.9%; the difference from control was
statistically significant (P = .003). These results
suggest that PKC phosphorylation is not absolutely required for
platelet aggregation but, if inhibited, leads to a delay in platelet
aggregation. These results also show that calpain function is essential
for platelet aggregation under our conditions of platelet preparation
and activation. Inhibition of adducin phosphorylation by PKC and
proteolysis by calpain could have a role in the observed inhibition of
platelet aggregation, although PKC and calpain have a large number of
substrates other than adducin.
Aggregation of platelets is a complex process comprising several steps:
secretion of granules, extension of filopodia and lamellipodia by
F-actin polymerization, and modulation of the integrin
We have shown that adducin was rapidly phosphorylated by PKC in platelets activated with thrombin, PMA, or calcium ionophore A23187, as defined by the use of a PKC site-specific phosphopeptide antibody. There are several possibilities for how PKC phosphorylation may regulate the function of adducin. Studies on the interaction of gamma adducin with PKC suggested that unphosphorylated gamma adducin can bind PKC, and, when PKC phosphorylates gamma adducin, it is released from its association.25 Further studies are needed to determine whether PKC and unphosphorylated adducin exhibit this interaction in platelets. Phosphorylation of recombinant alpha and beta adducin by PKC has been shown to decrease21 their interactions with spectrin and actin in vitro, and treatment of MDCK cells with PMA caused a redistribution of adducin from membrane to cytosol as seen by light microscopy.35 We have shown that adducin moves from high-speed pellet (membrane and cytoskeleton fraction) to supernatant (cytosolic fraction) during platelet activation and levels of PKC-phosphorylated adducin are much higher in the cytosol versus the pellet. These observations suggest that activation-induced PKC phosphorylation of adducin releases it from binding sites in the membrane skeleton or cytoskeleton. Erythrocyte adducin (alpha/beta) has been shown to cap actin filaments at the barbed end,18 and PKC phosphorylation reduces the F-actin capping activity of adducin.21 Platelets contain 0.5 µM F-actin barbed ends,36 based on measurements of average filament length and amount of polymerized actin per platelet.2 We have demonstrated that sufficient adducin is present (3-6 µM) to cap all actin filaments in the resting platelet. The concentration of adducin in platelets is similar to the 2 to 5 µM concentration of capping protein.36,37 However, the dissociation constant for binding to barbed ends is only 100 nmol/L for adducin18 versus 1 to 2 nmol/L for capping protein.38 In addition, it has not yet been demonstrated that platelet adducin (alpha/gamma) has actin-capping activity. Adducin is also a substrate for rho kinase, and phosphorylation by rho kinase, in contrast to phosphorylation by PKC, increases adducin's interaction with F-actin.22 Rho kinase is also activated during platelet activation,39 so it is not yet known how the combination of phosphorylation by PKC and rho kinase will affect adducin function in platelets. One important observation from our studies is that adducin was
proteolyzed during platelet activation. Inhibition of the proteolysis by calpeptin suggests that calpain is likely to be the protease involved. Calpain is an important component of platelet activation, causing highly specific calcium-induced proteolysis of several cytoskeleton proteins, including ABP-280, talin, spectrin, and cortactin.10,27,31 In addition, calpain proteolyzes the
Others have shown that inhibition of calpain blocks platelet granule
secretion, aggregation, and spreading on glass.41 Our results inhibiting proteolysis of adducin and platelet aggregation with
calpeptin are consistent with adducin having a role in platelet aggregation. Targeted deletion of the µ-calpain gene in mice
demonstrated that µ-calpain is crucial to normal platelet aggregation
and clot retraction.42 The proteolysis of ABP-280, talin,
and We have shown that adducin is crucial to the normal architecture of red blood cells.14 Targeted disruption of the beta adducin gene in mice caused a phenotype similar to the human disease hereditary spherocytosis. Platelets were unaffected in these mice because platelets do not express beta adducin. Future studies with targeted disruption of alpha and gamma adducin genes are likely to provide answers about the specific role of adducin in platelets.
We thank Laura Stewart for help with aggregation assays and Doug Bolgiano for help with statistical analyses.
Submitted July 28, 2001; accepted November 14, 2001.
Supported by grant R01-DK55005 from the National Institutes of Health, Bethesda, MD, and by a grant from the March of Dimes Birth Defects Foundation, Mamaroneck, NY.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Diana M. Gilligan, Puget Sound Blood Center, 921 Terry Ave, Seattle, WA 98104; e-mail: dianag{at}psbc.org.
1. White JG. Platelet secretion during clot retraction. Platelets. 2000;11:331-343[CrossRef][Medline] [Order article via Infotrieve].
2.
Hartwig JH.
Mechanisms of actin rearrangements mediating platelet activation.
J Cell Biol.
1992;118:1421-1442
3.
Shattil SJ.
Signaling through platelet integrin 4. Nachmias VT, Yoshida K-I. The cytoskeleton of the blood platelet: a dynamic structure. Adv Cell Biol. 1988;2:181-211. 5. Fox JEB. The platelet cytoskeleton. Thromb Haemost. 1993;70:884-893[Medline] [Order article via Infotrieve]. 6. Hartwig JH, Barkalow K, Azim A, Italiano J. The elegant platelet: signals controlling actin assembly. Thromb Haemost. 1999;82:392-398[Medline] [Order article via Infotrieve].
7.
Fox JEB, Boyles JK, Berndt MC, Steffen PK, Anderson LK.
Identification of a membrane skeleton in platelets.
J Cell Biol.
1988;106:1525-1538
8.
Hartwig JH, DeSisto M.
The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments.
J Cell Biol.
1991;112:407-425 9. Bennett V, Gilligan DM. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Ann Rev Cell Biol. 1993;9:27-66[CrossRef].
10.
Fox JEB, Reynolds CC, Morrow JS, Phillips DR.
Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation.
Blood.
1987;69:537-545
11.
Fox JEB.
Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes.
J Biol Chem.
1985;260:11970-11977
12.
Palfrey HC, Waseem A.
Protein kinase C in the human erythrocyte.
J Biol Chem.
1985;260:16021-16029
13.
Gardner K, Bennett V.
A new erythrocyte membrane-associated protein with calmodulin binding activity.
J Biol Chem.
1986;261:1339-1348
14.
Gilligan DM, Lozovatsky L, Gwynn B, Brugnara C, Narla M, Peters LL.
Targeted disruption of the beta adducin gene causes spherocytosis in mice.
Proc Natl Acad Sci U S A.
1999;96:10717-10722 15. Suriyapperuma S, Lozovatsky L, Ciciotte SL, Peters LL, Gilligan DM. The murine adducin gene family: alternative splicing and chromosomal localization. Mamm Genome. 2000;11:16-23[CrossRef][Medline] [Order article via Infotrieve].
16.
Hughes CA, Bennett V.
Adducin: a physical model with implications for function in assembly of spectrin-actin complexes.
J Biol Chem.
1995;270:18990-18996 17. Gardner K, Bennett V. Modulation of spectrin-actin assembly by erythrocyte adducin. Nature. 1987;328:359-362[CrossRef][Medline] [Order article via Infotrieve].
18.
Kuhlman PA, Hughes CA, Bennett V, Fowler VM.
A new function for adducin. Calcium/calmodulin regulated capping of the barbed ends of actin filaments.
J Biol Chem.
1996;271:7986-7991
19.
Mische SM, Mooseker MS, Morrow JS.
Erythrocyte adducin: a calmodulin-regulated actin-bundling protein that stimulates spectrin-actin binding.
J Cell Biol.
1987;105:2837-2845
20.
Matsuoka Y, Hughes CA, Bennett V.
Adducin regulation: definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C.
J Biol Chem.
1996;271:25157-25166
21.
Matsuoka S, Li X, Bennett V.
Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons.
J Cell Biol.
1998;142:485-497
22.
Kimura K, Fukata Y, Matsuoka Y, et al.
Regulation of the association of adducin with actin filaments by rho-associated kinase (rho-kinase) and myosin phosphatase.
J Biol Chem.
1998;273:5542-5548
23.
Fukata Y, Oshiro N, Kinoshita N, et al.
Phosphorylation of adducin by rho-kinase plays a crucial role in cell motility.
J Cell Biol.
1999;145:347-361
24.
Chapline C, Ramsay K, Klauck T, Jaken S.
Interaction cloning of protein kinase C substrates.
J Biol Chem.
1993;268:6858-6861
25.
Dong L, Chapline C, Mousseau B, et al.
35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.
J Biol Chem.
1995;270:25534-25540 26. Fox JEB, Reynolds CC, Boyles JK. Studying the platelet cytoskeleton in Triton X-100 lysates. Methods Enzymol. 1992;215:42-58[Medline] [Order article via Infotrieve].
27.
Fox JEB, Goll DE, Reynolds CC, Phillips DR.
Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation.
J Biol Chem.
1985;260:1060-1066 28. Siess W, Lapetina EG. Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C. Biochem J. 1988;255:309-318[Medline] [Order article via Infotrieve]. 29. Chen R, Liang N. Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA). Cell Biol Int. 1998;22:429-435[CrossRef][Medline] [Order article via Infotrieve].
30.
Zucker M, Troll W, Belman S.
The tumor-promoter phorbol ester (12-0-tetradecanoyl-phorbol-13-acetate), a potent aggregating agent for blood platelets.
J Cell Biol.
1974;60:325-336
31.
Huang C, Tandon NN, Greco NJ, Ni Y, Wang T, Zhan Xi.
Proteolysis of platelet cortactin by calpain.
J Biol Chem.
1997;272:19248-19252
32.
Du X, Saido TC, Tsubuki S, Indig FE, Williams MJ, Ginsberg MH.
Calpain cleavage of the cytoplasmic domain of the integrin b3 subunit.
J Biol Chem.
1995;270:26146-26151
33.
Schoenwaelder SM, Yuan Y, Jackson SP.
Calpain regulation of integrin 34. Tsujinaka T, Kajiwara Y, Kambayashi J, et al. Synthesis of a new cell penetrating calpain inhibitor (calpeptin). Biochem Biophys Res Comm. 1988;153:1201-1208[CrossRef][Medline] [Order article via Infotrieve].
35.
Kaiser HW, O'Keefe E, Bennett V.
Adducin: Ca2+-dependent association with sites of cell-cell contact.
J Cell Biol.
1989;109:557-569
36.
Barkalow K, Witke W, Kwiatkowski DJ, Hartwig JH.
Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein.
J Cell Biol.
1996;134:389-399 37. Nachmias VT, Golla R, Casella JF, Barron-Casella E. Cap Z, a calcium insensitive capping protein in resting and activated platelets. FEBS Lett. 1996;378:258-262[CrossRef][Medline] [Order article via Infotrieve]. 38. DiNubile MJ, Cassimeris L, Joyce M, Zigmond SH. Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-b2. Mol Biol Cell. 1995;6:1659-1671[Abstract]. 39. Fox JE. On the role of calpain and Rho proteins in regulating integrin-induced signaling. Thromb Haemost. 1999;82:385-391[Medline] [Order article via Infotrieve].
40.
Van de Water B, Tijdens IB, Verbrugge A, et al.
Cleavage of the actin-capping protein
41.
Croce K, Flaumenhaft R, Rivers M, et al.
Inhibition of calpain blocks platelet secretion, aggregation, and spreading.
J Biol Chem.
1999;274:36321-36327
42.
Azam M, Andrabi SS, Sahr KE, Kamath L, Kuliopulos A, Chishti AH.
Disruption of the mouse mu-calpain gene reveals an essential role in platelet function.
Mol Cell Biol.
2001;21:2213-2220
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  H. G. Chu, S. K. Weeks, D. M. Gilligan, and D. D. Rockey Host {alpha}-adducin is redistributed and localized to the inclusion membrane in Chlamydia- and Chlamydophila-infected cells Microbiology, December 1, 2008; 154(12): 3848 - 3855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. F. Robledo, S. L. Ciciotte, B. Gwynn, K. E. Sahr, D. M. Gilligan, N. Mohandas, and L. L. Peters Targeted deletion of {alpha}-adducin results in absent {beta}- and {gamma}-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice Blood, November 15, 2008; 112(10): 4298 - 4307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-L. Chen, Y.-T. Hsieh, and H.-C. Chen Phosphorylation of adducin by protein kinase C{delta} promotes cell motility J. Cell Sci., April 1, 2007; 120(7): 1157 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pula, K. Schuh, K. Nakayama, K. I. Nakayama, U. Walter, and A. W. Poole PKC{delta} regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation Blood, December 15, 2006; 108(13): 4035 - 4044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Kalfa, S. Pushkaran, N. Mohandas, J. H. Hartwig, V. M. Fowler, J. F. Johnson, C. H. Joiner, D. A. Williams, and Y. Zheng Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton Blood, December 1, 2006; 108(12): 3637 - 3645. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Rabenstein, N. A. Addy, B. J. Caldarone, Y. Asaka, L. M. Gruenbaum, L. L. Peters, D. M. Gilligan, R. M. Fitzsimonds, and M. R. Picciotto Impaired Synaptic Plasticity and Learning in Mice Lacking {beta}-Adducin, an Actin-Regulating Protein J. Neurosci., February 23, 2005; 25(8): 2138 - 2145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Lahortiga, J. L. Vizmanos, X. Agirre, I. Vazquez, J. C. Cigudosa, M. J. Larrayoz, F. Sala, A. Gorosquieta, K. Perez-Equiza, M. J. Calasanz, et al. NUP98 Is Fused to Adducin 3 in a Patient with T-Cell Acute Lymphoblastic Leukemia and Myeloid Markers, with a New Translocation t(10;11)(q25;p15) Cancer Res., June 15, 2003; 63(12): 3079 - 3083. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Barkalow, J. E. Italiano Jr., D. E. Chou, Y. Matsuoka, V. Bennett, and J. H. Hartwig {alpha}-Adducin dissociates from F-actin and spectrin during platelet activation J. Cell Biol., May 12, 2003; 161(3): 557 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Gruenbaum, D. M. Gilligan, M. R. Picciotto, S. Marinesco, and T. J. Carew Identification and Characterization of Aplysia Adducin, an Aplysia Cytoskeletal Protein Homologous to Mammalian Adducins: Increased Phosphorylation at a Protein Kinase C Consensus Site during Long-Term Synaptic Facilitation J. Neurosci., April 1, 2003; 23(7): 2675 - 2685. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
3 or GPIIbIIIa,
6 ng and 4.3 × 10
